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A tripartite paternally methylated region within the Gpr1-Zdbf2 imprinted domain on mouse chromosome 1 identified by meDIP-on-chip.

Hiura H, Sugawara A, Ogawa H, John RM, Miyauchi N, Miyanari Y, Horiike T, Li Y, Yaegashi N, Sasaki H, Kono T, Arima T - Nucleic Acids Res. (2010)

Bottom Line: In both humans and mice, the adjacent gene, Gpr1/GPR1, which encodes a G-protein-coupled receptor 1 protein with transmembrane domain, was also imprinted and paternally expressed.The Gpr1-Zdbf2 domain was most similar to the Rasgrf1 domain as both DNA methylation and the actively expressed allele were in cis on the paternal chromosome.This work demonstrates the effectiveness of meDIP-on-chip as a technique for identifying DMRs.

View Article: PubMed Central - PubMed

Affiliation: Innovation of New Biomedical Engineering Center, University of Tohoku, Aoba-ku, Sendai, Japan.

ABSTRACT
The parent-of-origin specific expression of imprinted genes relies on DNA methylation of CpG-dinucleotides at differentially methylated regions (DMRs) during gametogenesis. To date, four paternally methylated DMRs have been identified in screens based on conventional approaches. These DMRs are linked to the imprinted genes H19, Gtl2 (IG-DMR), Rasgrf1 and, most recently, Zdbf2 which encodes zinc finger, DBF-type containing 2. In this study, we applied a novel methylated-DNA immunoprecipitation-on-chip (meDIP-on-chip) method to genomic DNA from mouse parthenogenetic- and androgenetic-derived stem cells and sperm and identified 458 putative DMRs. This included the majority of known DMRs. We further characterized the paternally methylated Zdbf2/ZDBF2 DMR. In mice, this extensive germ line DMR spanned 16 kb and possessed an unusual tripartite structure. Methylation was dependent on DNA methyltransferase 3a (Dnmt3a), similar to H19 DMR and IG-DMR. In both humans and mice, the adjacent gene, Gpr1/GPR1, which encodes a G-protein-coupled receptor 1 protein with transmembrane domain, was also imprinted and paternally expressed. The Gpr1-Zdbf2 domain was most similar to the Rasgrf1 domain as both DNA methylation and the actively expressed allele were in cis on the paternal chromosome. This work demonstrates the effectiveness of meDIP-on-chip as a technique for identifying DMRs.

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Three paternally methylated DMRs on the Gpr1-Zdbf2 imprinted domain. (A) Physical map of the mouse Gpr1-Zdbf2 locus (upper panel). Black boxes represent the position of three paternally methylated DMRs, called DMR1 (5.0 kb), DMR2 (3.0 kb) and DMR3 (4.2 kb). Arrows above genes (white boxes) show the direction of transcription. White arrowheads indicate five times repeats of the 37 bp repetitive sequence. Close-up of the three paternally methylated DMRs (lower panel). The vertical bar represents a CpG site. The regions we analyzed bisulfite-PCR methylation sequencings were indicated 1–20. (B) Bisulfite-PCR sequencing results for four regions (regions 3, 7, 11 and 15) on genomic DNA prepared from sperm, MII oocytes, blastocysts from B6/JF1 mice and the kidney from B6/JF1 and reciprocal JF1/B6 mice. Each row represents a unique methylation profile within the pool of 20 clones sequenced. Closed and open circles represent methylated and unmethylated CpGs, respectively.
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Figure 2: Three paternally methylated DMRs on the Gpr1-Zdbf2 imprinted domain. (A) Physical map of the mouse Gpr1-Zdbf2 locus (upper panel). Black boxes represent the position of three paternally methylated DMRs, called DMR1 (5.0 kb), DMR2 (3.0 kb) and DMR3 (4.2 kb). Arrows above genes (white boxes) show the direction of transcription. White arrowheads indicate five times repeats of the 37 bp repetitive sequence. Close-up of the three paternally methylated DMRs (lower panel). The vertical bar represents a CpG site. The regions we analyzed bisulfite-PCR methylation sequencings were indicated 1–20. (B) Bisulfite-PCR sequencing results for four regions (regions 3, 7, 11 and 15) on genomic DNA prepared from sperm, MII oocytes, blastocysts from B6/JF1 mice and the kidney from B6/JF1 and reciprocal JF1/B6 mice. Each row represents a unique methylation profile within the pool of 20 clones sequenced. Closed and open circles represent methylated and unmethylated CpGs, respectively.

Mentions: We next performed meDIP-on-chip by applying the meDIP samples to mouse whole-genome tiling arrays. The fixed quantity value that had been obtained from this array analysis corrected the reference value. We looked for the regions under the following conditions: (i) at least three adjoined methylated probes (using neighborhood model supplied by Agilent Technologies Japan, p (Xbar) < 0.07) and (ii) a similar methylation pattern between AG-derived cells and sperm but dissimilar to PG-derived cells (normalized log ratio of the PG-derived cells probe < 0.5). We identified 458 candidate DMRs in the mouse genome. 141 were paternally methylated DMRs and 317 were maternally methylated DMRs (Figure 1, Tables 1 and 2). Of these, 20 were known DMRs. We correctly identified the IG-DMR (Gtl2) and Lit1 DMRs using the tiling arrays for mouse chromosome 7 and 12 (Figure 1B, upper panel). Using the tiling array for chromosome 1, we found the evidence of three closely linked paternally methylated DMRs (Figure 1B, lower panel) that lay within a 60 kb region between the imprinted Zdbf2 (zinc finger, DBF-type containing 2) gene and the uncharacterized gene, Gpr1 (G-protein-coupled receptor 1) (GenBank accession number NM146250) (Figure 2A). We had previously identified Zdbf2 as an imprinted gene linked to a DMR in a parallel study isolating imprinted genes based on their expression status (26). Not all the known DMRs were identified. The Ras/Grf1 DMR could not be identified because the sequence for this region had been excluded from the mouse tiling array due to its highly repetitive sequence. The H19 DMR was also not identified in the screen and this was most likely because the H19 DMR was methylated in both ADS and PDS material, as determined by COBRA, and therefore amplified from both meDIP samples. Nonetheless, these data on known DMRs indicated that meDIP would be an effective technique for identifying novel DMRs.Table 1.


A tripartite paternally methylated region within the Gpr1-Zdbf2 imprinted domain on mouse chromosome 1 identified by meDIP-on-chip.

Hiura H, Sugawara A, Ogawa H, John RM, Miyauchi N, Miyanari Y, Horiike T, Li Y, Yaegashi N, Sasaki H, Kono T, Arima T - Nucleic Acids Res. (2010)

Three paternally methylated DMRs on the Gpr1-Zdbf2 imprinted domain. (A) Physical map of the mouse Gpr1-Zdbf2 locus (upper panel). Black boxes represent the position of three paternally methylated DMRs, called DMR1 (5.0 kb), DMR2 (3.0 kb) and DMR3 (4.2 kb). Arrows above genes (white boxes) show the direction of transcription. White arrowheads indicate five times repeats of the 37 bp repetitive sequence. Close-up of the three paternally methylated DMRs (lower panel). The vertical bar represents a CpG site. The regions we analyzed bisulfite-PCR methylation sequencings were indicated 1–20. (B) Bisulfite-PCR sequencing results for four regions (regions 3, 7, 11 and 15) on genomic DNA prepared from sperm, MII oocytes, blastocysts from B6/JF1 mice and the kidney from B6/JF1 and reciprocal JF1/B6 mice. Each row represents a unique methylation profile within the pool of 20 clones sequenced. Closed and open circles represent methylated and unmethylated CpGs, respectively.
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Related In: Results  -  Collection

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Figure 2: Three paternally methylated DMRs on the Gpr1-Zdbf2 imprinted domain. (A) Physical map of the mouse Gpr1-Zdbf2 locus (upper panel). Black boxes represent the position of three paternally methylated DMRs, called DMR1 (5.0 kb), DMR2 (3.0 kb) and DMR3 (4.2 kb). Arrows above genes (white boxes) show the direction of transcription. White arrowheads indicate five times repeats of the 37 bp repetitive sequence. Close-up of the three paternally methylated DMRs (lower panel). The vertical bar represents a CpG site. The regions we analyzed bisulfite-PCR methylation sequencings were indicated 1–20. (B) Bisulfite-PCR sequencing results for four regions (regions 3, 7, 11 and 15) on genomic DNA prepared from sperm, MII oocytes, blastocysts from B6/JF1 mice and the kidney from B6/JF1 and reciprocal JF1/B6 mice. Each row represents a unique methylation profile within the pool of 20 clones sequenced. Closed and open circles represent methylated and unmethylated CpGs, respectively.
Mentions: We next performed meDIP-on-chip by applying the meDIP samples to mouse whole-genome tiling arrays. The fixed quantity value that had been obtained from this array analysis corrected the reference value. We looked for the regions under the following conditions: (i) at least three adjoined methylated probes (using neighborhood model supplied by Agilent Technologies Japan, p (Xbar) < 0.07) and (ii) a similar methylation pattern between AG-derived cells and sperm but dissimilar to PG-derived cells (normalized log ratio of the PG-derived cells probe < 0.5). We identified 458 candidate DMRs in the mouse genome. 141 were paternally methylated DMRs and 317 were maternally methylated DMRs (Figure 1, Tables 1 and 2). Of these, 20 were known DMRs. We correctly identified the IG-DMR (Gtl2) and Lit1 DMRs using the tiling arrays for mouse chromosome 7 and 12 (Figure 1B, upper panel). Using the tiling array for chromosome 1, we found the evidence of three closely linked paternally methylated DMRs (Figure 1B, lower panel) that lay within a 60 kb region between the imprinted Zdbf2 (zinc finger, DBF-type containing 2) gene and the uncharacterized gene, Gpr1 (G-protein-coupled receptor 1) (GenBank accession number NM146250) (Figure 2A). We had previously identified Zdbf2 as an imprinted gene linked to a DMR in a parallel study isolating imprinted genes based on their expression status (26). Not all the known DMRs were identified. The Ras/Grf1 DMR could not be identified because the sequence for this region had been excluded from the mouse tiling array due to its highly repetitive sequence. The H19 DMR was also not identified in the screen and this was most likely because the H19 DMR was methylated in both ADS and PDS material, as determined by COBRA, and therefore amplified from both meDIP samples. Nonetheless, these data on known DMRs indicated that meDIP would be an effective technique for identifying novel DMRs.Table 1.

Bottom Line: In both humans and mice, the adjacent gene, Gpr1/GPR1, which encodes a G-protein-coupled receptor 1 protein with transmembrane domain, was also imprinted and paternally expressed.The Gpr1-Zdbf2 domain was most similar to the Rasgrf1 domain as both DNA methylation and the actively expressed allele were in cis on the paternal chromosome.This work demonstrates the effectiveness of meDIP-on-chip as a technique for identifying DMRs.

View Article: PubMed Central - PubMed

Affiliation: Innovation of New Biomedical Engineering Center, University of Tohoku, Aoba-ku, Sendai, Japan.

ABSTRACT
The parent-of-origin specific expression of imprinted genes relies on DNA methylation of CpG-dinucleotides at differentially methylated regions (DMRs) during gametogenesis. To date, four paternally methylated DMRs have been identified in screens based on conventional approaches. These DMRs are linked to the imprinted genes H19, Gtl2 (IG-DMR), Rasgrf1 and, most recently, Zdbf2 which encodes zinc finger, DBF-type containing 2. In this study, we applied a novel methylated-DNA immunoprecipitation-on-chip (meDIP-on-chip) method to genomic DNA from mouse parthenogenetic- and androgenetic-derived stem cells and sperm and identified 458 putative DMRs. This included the majority of known DMRs. We further characterized the paternally methylated Zdbf2/ZDBF2 DMR. In mice, this extensive germ line DMR spanned 16 kb and possessed an unusual tripartite structure. Methylation was dependent on DNA methyltransferase 3a (Dnmt3a), similar to H19 DMR and IG-DMR. In both humans and mice, the adjacent gene, Gpr1/GPR1, which encodes a G-protein-coupled receptor 1 protein with transmembrane domain, was also imprinted and paternally expressed. The Gpr1-Zdbf2 domain was most similar to the Rasgrf1 domain as both DNA methylation and the actively expressed allele were in cis on the paternal chromosome. This work demonstrates the effectiveness of meDIP-on-chip as a technique for identifying DMRs.

Show MeSH
Related in: MedlinePlus